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Aortic inhibition of the JNK isoform 2 after Roux‐en‐Y gastric bypass was associated with improved insulin and glucagon‐like peptide‐1–mediated endothelium‐dependent vasorelaxation as a result of preserved NO bioavailability and blunted oxidative stress.

What Are the Clinical Implications?

Our data suggest that novel drugs that selectively blunt JNK2 activation without the downsides of available JNK inhibitors may become a promising therapeutic option against obesity‐induced endothelial dysfunction and its associated cardiovascular disease.

It will be important to assess the vascular and metabolic effects of a combination therapy of Roux‐en‐Y gastric bypass and JNK inhibition.

It is intriguing to hypothesize that, as a result of such dual treatment, a higher increase in circulating glucagon‐like peptide‐1 and/or in other gut hormones may synergistically potentiate the benefits observed in the present experiments.

Introduction

Obesity is associated with comorbidities, such as type 2 diabetes mellitus and dyslipidemia, which increase cardiovascular risk and mortality.1, 2 In obesity, a systemic chronic inflammation leads to oxidative stress, insulin resistance, and endothelial dysfunction.3, 4 Bariatric surgery, in particular Roux‐en‐Y gastric bypass (RYGB), is the most effective treatment against obesity.1 RYGB achieves sustained weight loss and significant long‐term improvement of comorbidities; it also reduces cardiovascular morbidity and mortality.1, 5, 6 In addition to weight loss, surgery‐specific changes of gut hormones, like glucagon‐like peptide‐1 (GLP‐1) and bile acids, may contribute to these improvements.7, 8, 9

The c‐Jun N‐terminal kinase (JNK) family of proteins links obesity with insulin resistance and endothelial dysfunction. Of the 3 known JNK isoforms, JNK1 and JNK2 are expressed ubiquitously, whereas JNK3 expression is restricted to the brain, heart, and testes.10 On activation, JNK suppresses cellular insulin signaling via inhibitory phosphorylation of insulin receptor substrate‐1 (IRS‐1) at Ser307 (Ser307–IRS‐1).11 In obese mice, whole‐body deletion of JNK1 (JNK1−/−), but not of JNK 2 (JNK2−/−), decreased total JNK activity in liver, muscle, and adipose tissue, and reduced hepatic Ser307–IRS‐1 phosphorylation.12 This blunted weight gain and improved glucose tolerance and insulin sensitivity.12 The deletion of JNK2, performed either globally or specifically in the liver, was also associated with the same favorable metabolic profile13, 14 previously attributed to JNK1 deletion. Hence, the in vivo tissue‐specific interactions between the 2 JNK isoforms deserve further investigation under physiological and pathological conditions.12, 13, 14

JNK is also critical in the pathophysiology of cardiovascular disease. JNK2−/− mice are protected from hypercholesterolemia‐induced oxidative stress, endothelial dysfunction,15 and atherosclerosis.16 JNK2 may inhibit endothelial NO synthase (eNOS) by phosphorylation at Ser116 (Ser116‐eNOS).17, 18 Total JNK activation and Ser307–IRS‐1 phosphorylation, which are increased in the aorta of spontaneously hypertensive rats, are associated with vascular insulin resistance.19 Furthermore, in rat aortic rings, the JNK inhibitor SP60012520 promotes vasodilation.21 However, SP600125 is not JNK specific and targets other kinases.22, 23 This drawback prompted the development of more JNK‐specific inhibitors (eg, the peptide inhibitor D‐JNKi‐1 [D‐JNK]), which more selectively blocks all JNK isoforms.24 Two‐week treatment of obese diabetic db/db mice with the l‐enantiomer of D‐JNK improves glucose tolerance and insulin sensitivity, increases protein kinase B (Akt) activation, and decreases Ser307–IRS‐1 phosphorylation in liver, fat, and muscle.25 Moreover, D‐JNK treatment exerts neuroprotective effects in mouse models of cerebral ischemia, and the compound is in preclinical trials.26

RYGB decreased total JNK activation and Ser307–IRS‐1 inhibitory phosphorylation in the liver of type 2 diabetic rats 2 weeks after surgery.27 We previously showed, in obese rats, a rapid improvement of endothelial function after RYGB.8 In the aorta, RYGB decreased total JNK phosphorylation and oxidative stress and activated the Akt/eNOS signaling pathway, improving NO bioavailability, independent of body weight loss.8 Therefore, the present study tested if pharmacological JNK inhibition in vivo in obese rats mimics the protective endothelial effects of RYGB. Moreover, the specific contribution of vascular JNK1 versus JNK2 in obesity‐induced endothelial dysfunction and in its improvement after RYGB was investigated.

Animals and Experimental Procedures

Male Wistar rats were fed chow (lean non–operated on age‐matched controls, n=6) or a high‐fat high‐cholesterol diet to induce obesity (60% kcal fat and 1.25% cholesterol; n=46) for 7 weeks before and after surgery.8 Obese rats were randomized to RYGB or sham surgery, and all procedures were performed as described.8 Treatments started on the day after surgery. A pilot (proof‐of‐concept) study and a main experiment were performed with the 2 JNK inhibitors most commonly used for in vivo investigations.20, 24 In the pilot study, sham–operated rats received SP600125, 40 mg/kg per day SC (SP600125, n=8), or vehicle dimethyl sulfoxide sc (controlDMSO, n=6). The main experiment compared RYGB (n=10) with sham–operated ad libitum–fed rats treated for 8 days with either the selective JNK inhibitor D‐JNK (once‐daily sc injection of 20 mg/kg per day; H‐dqsrpvqpflnlttprkprpprrrqrrkkrG‐NH2; n=13) or an equimolar dose (8.3 mg/kg) of the control peptide D‐TAT sc (controlD‐TAT; H‐pprrrqrrkkrG‐NH2; n=9). Some controlD‐TAT and D‐JNK rats were kept in metabolic cages to measure water intake and urine excretion. The Cantonal Veterinary Office of Zurich (Zurich, Switzerland) approved all animal experiments (approval No. 060/2015_860).

Organ Chamber Experiments

The thoracic aorta was cut into 2‐ to 3‐mm‐long rings and connected to an isometric force transducer (Multi‐Myograph 610M; Danish Myo Technology A/S, Aarhus, Denmark), suspended in an organ chamber filled with 6 mL Krebs‐Ringer bicarbonate solution (37°C, pH 7.4, 95% O2, 5% CO2). Isometric tension was recorded continuously, as previously described.8, 15 After a 30‐minute equilibration period, rings were gradually stretched to reach the optimal point of their length‐tension curve, as determined by the contraction in response to potassium chloride (100 mmol/L). During submaximal contraction to norepinephrine (3×10−6 mol/L), concentration‐relaxation curves were obtained in a cumulative manner in response to insulin (10−11 to 10−5 mol/L) and GLP‐1 (7‐36) amide (10−12 to 10−6 mol/L).28, 29 Responses to insulin and GLP‐1 were recorded in the presence or absence of Nω‐nitro‐l‐arginine methyl ester (10−4 mol/L), a nonselective NO synthase inhibitor, or of the free radical scavenger polyethylene glycol–superoxide dismutase (150 U/mL). To study the direct vascular effects of D‐JNK, a subset of aortic rings were incubated ex vivo for 30 minutes with 5 μmol/L D‐JNK before insulin or GLP‐1. The NO donor sodium nitroprusside (10−10 to 10−5 mol/L) was added to test endothelium‐independent vasodilation. Relaxations were expressed as a percentage of the precontraction with norepinephrine.

In Vitro Assays

Statistical Analysis

Quantitative data are presented as mean±SD or median with interquartile range. Statistical analysis was conducted with unpaired Student t test or by 1‐way ANOVA, followed by the Bonferroni post hoc test, as appropriate. Two‐way ANOVA with repeated measures was used to compare repeated measurements in the same animals. Data were subjected to the statistical analysis after a test of the data distribution status using the Kolmogorov‐Smirnov test. If the homogeneity of variance assumption was violated, the nonparametric Kruskal‐Wallis test was used instead.

All tests were 2 sided, and statistical significance was accepted if the null hypothesis could be rejected at P<0.05. All analyses were performed with GraphPad Prism Software (version 7).

Results

Aortic relaxation in response to GLP‐1 and insulin improved similarly after D‐JNK and RYGB when compared with obese controlD‐TAT animals (Figure 1A and ​and1B).1B). Vasodilation was abolished by preincubation with the eNOS inhibitor Nω‐nitro‐l‐arginine methyl ester, indicating an endothelial NO‐dependent mechanism (Figure S1A and S1B). Beforehand, we showed that in lean non–operated age‐matched and chow‐fed male Wistar rats, D‐JNK did not alter GLP‐1's and insulin's baseline vasodilatory effects compared with vehicle‐treated controls (Figure S1C and S1D). Results after D‐JNK treatment strengthened the findings of the pilot experiment (Figure S1E and S1F), in which rats receiving the JNK inhibitor SP600125 also improved vascular relaxation, although the effect was less pronounced than after RYGB.

Cumulative concentration‐response curves of aortic rings isolated 8 days after Roux‐en‐Y gastric bypass (RYGB) or sham surgery/start of treatment after submaximal contraction to norepinephrine in response to glucagon‐like...

To test whether the improved relaxation seen in D‐JNK rats resulted from the direct vascular benefits of JNK inhibition, a subset of aortic rings isolated from D‐JNK and controlD‐TAT rats were preincubated with 5 μmol/L D‐JNK for 30 minutes ex vivo21, 24 before organ chamber experiments. Insulin‐ and GLP‐1–induced relaxations improved in controlD‐TAT rings treated ex vivo with D‐JNK compared with untreated rings, suggesting a direct aortic action of D‐JNK (Figure 1C and ​and1D).1D). By contrast, ex vivo D‐JNK preincubation did not further ameliorate the response of aortas from the rats that had received D‐JNK for 8 days (Figure 1E and ​and11F).

Next, we assessed the specific contribution of the 2 vascular JNK isoforms in obesity‐induced endothelial dysfunction. Phosphorylation of JNK2, but not of JNK1, increased in aortas of obese controlD‐TAT when compared with chow‐fed lean D‐TAT rats (Figure 2A). Furthermore, aortic JNK2 phosphorylation was significantly decreased in RYGB and D‐JNK–treated rats compared with obese controlD‐TAT rats, whereas JNK1 phosphorylation was not affected (Figure 2B). A similar result was obtained after the pilot treatment with SP600125 (Figure S2A).

The vascular Akt/eNOS signaling, which is activated by insulin and GLP‐18, 28 and inhibited by JNK,11, 17 was assessed by Western blotting in aortic lysates. The JNK‐dependent inhibitory Ser307–IRS‐1 phosphorylation decreased comparably after RYGB and D‐JNK (Figure 2C), whereas the activatory Akt‐Ser473 phosphorylation increased (Figure 2D). This led to enhanced phosphorylation of the activatory eNOS‐Ser1177 (Figure 3A), a favorable dimer/monomer ratio of eNOS (Figure 3B), which is essential for the proper coupling and function of the enzyme.30, 31 Finally, it augments NO production, as assessed by increased aortic cGMP levels (Figure 3C). In vivo SP600125 treatment also protected the insulin‐ and GLP‐1–dependent vascular signaling pathway, leading to preserved NO bioavailability (Figure S2B through S2E). The phosphorylation of Ser116 is a putative JNK2 target for the inhibition of eNOS.17, 18 However, its assessment was inconclusive because of lack of specificity of the commercially available anti–phosphorylated Ser116‐eNOS antibody in rat aortic tissue (Figure S3). Additional players able to modulate vascular insulin resistance,32, 33, 34 such as PKC (α/β, β II, and δ; Figure S4A through S4C) and extracellular signal regulated kinase 1/2 (p44 and p42; Figure S4E), were assessed. No difference in phosphorylation was observed between controls and rats after RYGB or JNK inhibition, which further supports the role of reduced aortic JNK2 phosphorylation as a crucial mediator of the vascular benefits of RYGB. Only the phosphorylation of p38 MAPK was increased on JNK inhibition and unchanged in control and RYGB rats (Figure S4D), which may be a potential off‐target effect of long‐term systemic in vivo D‐JNK treatment.

JNK Inhibition Reduces Vascular Oxidative Stress Similar to RYGB

JNK enhances vascular oxidative stress, which, in turn, reduces NO bioavailability and leads to endothelial dysfunction.35, 36 Decreased oxidative stress after RYGB protects endothelial function.8 Therefore, it was further assessed if in vivo JNK inhibition also mimics the effect of RYGB on the vascular reduction‐oxidation balance. Preincubation with the free radical scavenger superoxide dismutase improved relaxation in response to insulin and GLP‐1 only in aortic rings of controlD‐TAT rats. No further improvements were observed in RYGB‐ and D‐JNK–treated rats, which suggests markedly reduced oxidative stress in these contexts (Figure 3D and ​and3E).3E). Concurrent with this notion, dihydroethidium staining of aortic sections showed decreased superoxide anion (O2−) levels after RYGB and after D‐JNK treatment (Figure 3F). NADPH oxidase activity, which contributes to the generation of O2−,35 was also significantly decreased in the aortas of both RYGB and D‐JNK rats, thus contributing to preserved NO bioavailability (Figure 4A). Similar results were obtained after SP600125 treatment (Figure S5A and S5B).

GLP‐1 Contributes to Vascular JNK2 Inhibition After RYGB

Increased circulating GLP‐1 contributes to the beneficial cardiovascular effects of RYGB.8, 9 GLP‐1–dependent PKA activation inhibits JNK1 and JNK2 in various tissues, including the endothelium,34, 37 where it also activates the Akt/eNOS pathway.38, 39 In line with our previous results,8 fasting plasma GLP‐1 levels were increased after RYGB (Figure 4B). Surprisingly, D‐JNK treatment also increased GLP‐1 levels compared with controlD‐TAT rats, although to a lesser extent than RYGB (Figure 4B). By contrast, SP600125 did not affect circulating GLP‐1 levels (Figure S5C). The aortic expression of the GLP‐1 receptor was up‐regulated in RYGB and D‐JNK rats. This was accompanied downstream by enhanced phosphorylation of the PKA isoform C‐α at Thr197 and of the cAMP response element binding protein at Ser133 (Figure 4C through ​through4E),4E), which lie upstream of JNK1 and JNK2 inhibition.34, 37 To further ascertain the role of GLP‐1 in modulating JNK2 phosphorylation after RYGB, we analyzed aortas from our previous study, in which sham–operated on rats were treated with the GLP‐1 analogue liraglutide and RYGB rats received the GLP‐1 receptor antagonist exendin‐9 for 8 days after surgery.8 JNK2 phosphorylation remained high in RYGB rats on GLP‐1 receptor blockade, whereas liraglutide treatment in sham–operated on rats decreased JNK2 phosphorylation. JNK1 phosphorylation was unchanged in all groups (Figure 5A). These results suggest that vascular JNK2 inactivation may contribute to the GLP‐1–dependent improvement of endothelial dysfunction seen after RYGB.8 In line with previous results,8 RYGB reduced food intake and body weight (Figure 5B and ​and5C).5C). D‐JNK treatment affected food intake and body weight similar to RYGB (Figure 5B and ​and5C),5C), whereas SP600125 had no effect on these 2 parameters compared with its vehicle (Figure S6A and S6B). Non–operated chow‐fed rats treated for 8 days with D‐TAT and D‐JNK did not show differences in body weight between the 2 groups (Figure S6C). This difference between the 2 JNK inhibitors prompted additional investigations to assess potential toxicity associated with the pharmacological treatment (Figure S7), as described in Data S1.

Studies using tissue‐specific gene targeting in mice pointed initially to JNK112, 14 and later to JNK213 as pivotal players in the pathophysiological characteristics of obesity and type 2 diabetes mellitus. Hence, the role of the 2 isoforms in the overall JNK activity in metabolic disease is yet unclear. JNK2−/− mice were protected against hypercholesterolemia‐induced endothelial dysfunction15 and atheroma formation,16 which may suggest a specific contribution of JNK isoform 2 in the pathophysiological characteristics of atherosclerotic disease. We and others described also the association between total JNK activation and endothelial dysfunction induced by obesity8, 40 and type 2 diabetes mellitus41; however, the role of JNK1 versus JNK2 has not yet been elucidated in this context.

Our study shows that in obesity, enhanced aortic‐specific JNK2 phosphorylation impairs the insulin/IRS‐1– and GLP‐1/GLP‐1 receptor–dependent Akt/eNOS/NO pathway, leading to endothelial dysfunction, whereas JNK1 phosphorylation remains unchanged. Indeed, RYGB reduces JNK2 hyperphosphorylation and, thus, preserves the common insulin‐ and GLP‐1–mediated Akt/eNOS‐dependent NO production and restores endothelial function.29 RYGB‐mediated specific JNK2 inhibition was associated with decreased aortic Ser307–IRS‐1 phosphorylation. To date, the inhibitory Ser307–IRS‐1 phosphorylation has been linked to total JNK activity in the context of hepatic insulin resistance.12 Only one study in spontaneously hypertensive rats linked Ser307–IRS‐1 phosphorylation to aortic insulin resistance as a result of total JNK increased activity, with no further investigations on JNK isoform specificity.19

In Vivo Inhibition of JNK Mimics the Endothelial Protective Effects of RYGB

In vivo treatment with 2 different JNK inhibitors improved endothelium‐mediated vasodilation and decreased aortic JNK2 phosphorylation. Activation of the intracellular Akt/eNOS signaling led to higher NO production, thus mimicking the effect of RYGB. D‐JNK treatment decreased weight loss and food intake equally to RYGB; however, body weight loss per se is not sufficient to improve endothelial function, as previously shown in sham–operated rats whose body weight loss was matched to RYGB by food restriction.8 We observed that in rats that were body weight matched to RYGB rats, endothelial dysfunction persisted and was associated with increased total JNK phosphorylation at the aortic level.8 Furthermore, the pilot study showed that the favorable impact of SP600125 on endothelial function can also be achieved in the absence of body weight loss or reduced eating, suggesting a specific vascular benefit on pharmacological JNK inhibition. In addition, short‐term ex vivo D‐JNK treatment improved vasodilation of aortas of controlD‐TAT rats but had no incremental benefit in animals receiving long‐term in vivo D‐JNK treatment. These findings support a JNK inhibitory effect directly exerted in the aortas to complement other systemic beneficial metabolic effects of in vivo D‐JNK treatment.25, 42

JNKs promote reactive oxygen species production, which impairs NO bioavailability and leads to endothelial dysfunction.15, 36 In agreement with our previous findings,8 the preincubation of aortic rings with the radical scavenger superoxide dismutase improved insulin‐ and GLP‐1–mediated relaxations of aortas in controlD‐TAT rats but not in RYGB and D‐JNK treated rats. This result confirms the reduction of vascular oxidative stress, as shown by the reduced O2− levels and NADPH oxidase activity,35 in the aortas of RYGB and D‐JNK rats. In microvascular endothelial cells, NADPH‐dependent increase of reactive oxygen species after hypoxia/reoxygenation was blunted by SP600125 and by dominant‐negative JNK1, whereas the role of JNK2 in that condition had not been investigated in detail.43 Our findings suggest that in the aorta, obesity may specifically induce NADPH oxidase via JNK2, rather than JNK1, activation.

Finally, the present study focused on the role of upstream GLP‐1–dependent molecular pathways in mediating aortic JNK2 inactivation after RYGB. In line with our previous results,8 fasting plasma GLP‐1 levels and aortic GLP‐1 receptor expression were increased after RYGB. GLP‐1–dependent aortic PKA Thr197 and cAMP response element binding protein Ser133 activatory phosphorylations were increased and accompanied by downstream JNK2 inhibition, thus strengthening the mechanistic link between increased GLP‐1 signaling after RYGB and aortic JNK2 inhibition. This notion was supported by the fact that in vivo GLP‐1 receptor antagonism with exendin‐9 in RYGB rats blocked the JNK2 inhibition achieved by RYGB, whereas GLP‐1 agonism with liraglutide tended to mimic the benefit of RYGB in obese sham–operated on rats.

Interestingly, systemic administration of D‐JNK also increased circulating GLP‐1 levels; this was not observed with SP600125, possibly because of its lower potency and specificity.23 Peripheral GLP‐1 is secreted from intestinal endocrine cells in response to nutrients44 and insulin.45 Therefore, JNK inhibition in the small intestine may influence GLP‐1 secretion through the insulin signaling. Hence, systemic JNK inhibition may improve endothelium‐mediated vasodilation through a direct inactivation of vascular JNK2, and indirectly through increased GLP‐1 signaling, which may further contribute to blunt JNK2 and to activate eNOS in aortas, as summarized in Figure 6. In future studies, it would, therefore, be important to assess the vascular and metabolic effects of a combination therapy between RYGB and JNK inhibition in comparison to the single treatment reported in the present study. It is intriguing to hypothesize that, as a result of such dual treatment, higher increases in circulating GLP‐1 and/or in other gut hormones may synergistically potentiate the benefits observed in the present experiments.

Study Limitations

Reduced JNK2‐dependent inhibitory phosphorylated Ser116‐eNOS may have contributed to the observed benefits of RYGB and JNK inhibition,17, 18 but lack of specificity of the commercially available antibodies in the rat aorta did not allow a satisfactory assessment of this putative JNK2 target.

SP600125 inhibits multiple other kinases besides JNK,23 which limits its clinical application. On the other hand, D‐JNK treatment, despite a high JNK specificity,24 may induce renal toxicity, in particular in obese animals, which seems attributable to its carrier peptide, D‐TAT, and thus is independent of the D‐JNK inhibitory activity, as previously shown.46 Indeed, we observed proximal tubule injury in the carrier peptide–treated controlD‐TAT and in D‐JNK rats. In a previous study, despite improved glucose control, albuminuria worsened after in vivo treatment with the peptide JNK inhibitor in diabetic db/db mice42 by an effect possibly linked to the D‐TAT carrier peptide–induced kidney toxicity, independent of the metabolic benefits of JNK inhibition.42, 46 In our study, SP600125 treatment was not associated with kidney injury, further supporting the presence of JNK‐independent cytotoxicity (ie, toxicity related to the D‐TAT carrier peptide).46 Last, in D‐JNK treated rats, aortic phosphorylation of p38 MAPK was increased, in contrast to previous in vitro studies testing D‐JNK specificity.26, 47 Although potential off‐target effects of long‐term systemic in vivo D‐JNK administration cannot be excluded, in our setting, this treatment led to overall improved aortic Akt/eNOS signaling and NO bioavailability.

Overall, our data suggest that novel drugs, able to selectively blunt JNK2 activation without the downsides of available JNK inhibitors, may become a promising therapeutic option against obesity‐induced endothelial dysfunction and its associated cardiovascular disease.

Conclusions

Obesity‐induced activation of vascular JNK2 and the associated endothelial dysfunction were reversed by RYGB, through GLP‐1–dependent signaling. These beneficial effects of RYGB were mimicked by in vivo pharmacological JNK inhibition, highlighting the potential therapeutic relevance of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity.

Author Contributions

The contributions to the article were as follows: study design: Osto and Lutz; study conduct, data collection, and analysis: Osto, Doytcheva, Bächler, Tarasco, Marzolla, Engeli, Pellegrini, Stivala, and Rohrer; writing: Osto, Lutz, Doytcheva, Marzolla, Tona, Camici, Vanhoutte, Matter, and Lüscher. All authors had full access and take full responsibility for the integrity of the data and the accuracy of the data analysis.

Sources of Funding

This work was supported, in part, by the University of Zurich Forschungskredit (FK‐85701‐02‐01 to Osto and FK‐15‐051 to Doytcheva); the Swiss National Science Foundation Ambizione Grant (PZOOP3_161S0S/1 to Osto); the Swiss Card‐Onco‐Grant, Alfred and Annemarie von Sick Grant (Lutz and Osto); and the Swiss Heart Foundation (Lutz and Osto).

Figure S7. Histological analysis by hematoxylin and eosin (H&E) staining of kidney from (A) a D‐JNK and (B) a controlsD‐TAT rat euthanized 8 days after sham surgery/start of treatment. Cumulative 24‐hour urine excretion in (C) controlsD‐TAT rats and (D) peptide inhibitor D‐JNKi‐1 (D‐JNK) rats, and cumulative 24‐hour water intake in (E) controlsD‐TAT rats and (F) D‐JNK rats 1 day before and 7 and 8 days after surgery and start of treatment (#, ##, ###P<0.05, P<0.01, P<0.001 Day ‐1 vs Day 7 and Day 8, as determined by one‐way ANOVA and Bonferroni post hoc test; n=6 per group). qPCR analysis of kidney expression of (G) kidney injury molecule‐1 (KIM‐1) and (H) neutrophil gelatinase‐associated lipocalin (NGAL) 8 days after surgery and start of treatment (°, °°°P<0.05, P<0.001 D‐JNK vs all other groups, as determined by Kruskal–Wallis test and Dunn's Multiple Comparison test; n=4–7 per group). Results are shown as mean±SD except in (G and H) where are presented as median±IQR.